Genes coding for metabolic pathway proteins

ABSTRACT

The invention relates to novel nucleic acid molecules, to the use thereof for constructing genetically improved microorganisms and to methods for preparing fine chemicals, in particular amino acids, with the aid of said genetically improved microorganisms.

BACKGROUND OF THE INVENTION

Particular products and byproducts of naturally occurring metabolicprocesses in cells are used in many branches of industry, including thefood industry, the animal feed industry, the cosmetics industry and thepharmaceutical industry. These molecules which are collectively referredto as “fine chemicals” comprise organic acids, both proteinogenic andnonproteinogenic amino acids, nucleotides and nucleosides, lipids andfatty acids, diols, carbohydrates, aromatic compounds, vitamins,cofactors and enzymes. They are best produced by means of cultivating,on a large scale, bacteria which have been developed to produce andsecrete large amounts of one or more molecules of interest. An organismwhich is particularly suitable for this purpose is Corynebacteriumglutamicum, a Gram-positive nonpathogenic bacterium. Using strainselection, a number of mutant strains have been developed which producevarious desirable compounds. The selection of strains which are improvedwith respect to the production of a particular molecule is, however, atime-consuming and difficult process.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides novel nucleic acid molecules which can beused for identifying or classifying Corynebacterium glutamicum orrelated bacteria species. C. glutamicum is a Gram-positive aerobicbacterium which is widely used in industry for the large-scaleproduction of a number of fine chemicals and also for the degradation ofhydrocarbons (e.g. in the case of crude oil spills) and for theoxidation of terpenoids. The nucleic acid molecules may therefore beused for identifying microorganisms which can be used for producing finechemicals, for example by fermentation processes. Although C. glutamicumitself is nonpathogenic, it is, however, related to otherCorynebacterium species such as Corynebacterium diphtheriae (thediphtheria pathogen), which are major pathogens in humans. The abilityto identify the presence of Corynebacterium species may therefore alsobe of significant clinical importance, for example in diagnosticapplications. Moreover, said nucleic acid molecules may serve asreference points for mapping the C. glutamicum genome or the genomes ofrelated organisms.

These novel nucleic acid molecules encode proteins which are referred toherein as metabolic pathway (MP) proteins. These MP proteins may, forexample, exert a function which is involved in the transcriptional,translational or posttranslational regulation of proteins which arecrucial for the normal metabolic functioning of cells. Owing to theavailability of cloning vectors for use in Corynebacterium glutamicum,as disclosed, for example, in Sinskey et al., U.S. Pat. No. 4,649,119,and of techniques for the genetic manipulation of C. glutamicum and therelated Brevibacterium species (e.g. lactofermentum) Yoshihama et al.,J. Bacteriol. 162 (1985) 591-597; Katsumata et al., J. Bacteriol. 159(1984) 306-311; and Santamaria et al. J. Gen. Microbiol. 130 (1984)2237-2246), the nucleic acid molecules of the invention can be used forgenetic manipulation of said organism in order to make it a better andmore efficient producer of one or more fine chemicals.

The improved yield, production and/or efficiency of production of a finechemical may be caused directly or indirectly by manipulating a gene ofthe invention. More specifically, modifications in C. glutamicum MPproteins which usually regulate the yield, production and/or efficiencyof production of a fine chemical of a fine-chemical metabolic pathwaymay may have a direct effect on the total production or production rateof one or more of these desired compounds from said organism.Modifications in those proteins which are involved in these metabolicpathways may also have an indirect effect on the yield, productionand/or efficiency of production of a desired fine chemical. Metabolicregulation is inevitably complex and the regulatory mechanisms whicheffect the different pathways may overlap in many places so that morethan one metabolic pathway can be adjusted quickly according to aparticular cellular event. This makes it possible for the modificationof a regulatory protein for one metabolic pathway to also affect manyother metabolic pathways, some of which may be involved in thebiosynthesis or degradation of a fine chemical of interest. In thisindirect manner, the modulation of the action of an MP protein may havean effect on the production of a fine chemical which is produced via ametabolic pathway different from that directly regulated by said MPprotein.

The nucleic acid and protein molecules of the invention may be used inorder to directly improve the yield, production and/or efficiency ofproduction of one or more fine chemicals of interest fromCorynebacterium glutamicum. It is possible, by means of generecombination techniques known in the art, to manipulate one or moreregulatory proteins of the invention such that their functions aremodulated. The mutation of an MP protein which is involved in repressingthe transcription of a gene encoding an enzyme required for thebiosynthesis of an amino acid, such that said acid is no longer capableof repressing the transcription, may cause, for example, an increase inthe production of said amino acid. Accordingly, modification of theactivity of an MP protein, which causes an increased translation oractivates posttranslational modification of a C. glutamicum proteininvolved in the biosynthesis of a fine chemical of interest, may in turnincrease the production of said chemical. The opposite situation maylikewise be useful: by increasing the repression of transcription ortranslation or by posttranslational negative modification of a C.glutamicum protein involved in regulating the degradation pathway of acompound, it is possible to increase the production of said chemical. Inany case, the total yield or the production rate of the fine chemical ofinterest may be increased.

Likewise, it is possible that said modifications in the protein andnucleotide molecules of the invention may improve the yield, productionand/or efficiency of production of fine chemicals via indirectmechanisms. The metabolism of a particular compound is inevitably linkedto other biosynthetic and degradation pathways in the cell and necessarycofactors, intermediates or substrates of a metabolic pathway areprobably provided or limited by another metabolic pathway. Modulatingone or more regulatory proteins of the invention can therefore influencethe efficiency of the activity of other biosynthetic or degradationpathways of fine chemicals. In addition to this, the manipulation of oneor more regulatory proteins may increase the overall ability of the cellto grow and to propagate in culture, particularly in large-scalefermentation cultures in which the growth conditions may be suboptimal.It is possible to increase the biosynthesis of nucleosides and possiblycell division, for example by mutating an inventive MP protein whichusually a repression of the biosynthesis of nucleosides as a reaction toa suboptimal extracellular supply of nutrients (thereby preventing celldivision), such that said protein has a lower repressor activity.Modifications in those MP proteins which cause increased cell growth andincreased cell division in culture may cause an increase in the yield,production and/or efficiency of production of one or more fine chemicalsof interest from the culture, at least owing to the increased number ofcells producing said chemical in culture.

The invention provides novel nucleic acid molecules encoding proteinswhich are referred to here as metabolic regulatory (MP) proteins andwhich are, for example, capable of carrying out an enzymic step which isinvolved in the transcriptional, translational or posttranslationalregulation of metabolic pathways in C. glutamicum. Nucleic acidmolecules which encode an MP protein are referred to here as MP nucleicacid molecules. In a preferred embodiment, the MP protein is involved inthe transcriptional, translational or posttranslational regulation ofone or more metabolic pathways. Examples of such proteins are thoseencoded by the genes listed in Table 1.

Consequently, one aspect of the invention relates to isolated nucleicacid molecules (e.g. cDNAs) comprising a nucleotide sequence whichencodes an MP protein or biologically active sections thereof andnucleic acid fragments which are suitable as primers or hybridizationprobes for detecting or amplifying MP-encoding nucleic acid (e.g. DNA ormRNA). In particularly preferred embodiments, the isolated nucleic acidmolecule comprises any of the nucleotide sequences listed in Appendix Aor the coding region or a complement thereof of any of these nucleotidesequences. In other preferred embodiments, the isolated nucleic acidmolecule encodes any of the amino acid sequences listed in Appendix B.The preferred MP proteins of the invention likewise have preferably atleast one of the MP activities described herein.

Appendix A defines hereinbelow the nucleic acid sequences of thesequence listing together with the sequence modifications at therelevant position, described in Table 1.

Appendix B defines hereinbelow the polypeptide sequences of the sequencelisting together with the sequence modifications at the relevantposition, described in Table 1.

In a further embodiment, the isolated nucleic acid molecule is at least15 nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule which comprises a nucleotide sequence of AppendixA. The isolated nucleic acid molecule preferably corresponds to anaturally occurring nucleic acid molecule. The isolated nucleic acidmore preferably encodes a naturally occurring C. glutamicum MP proteinor a biologically active section thereof.

Another aspect of the invention relates to vectors, for examplerecombinant expression vectors, which contain the nucleic acid moleculesof the invention and to host cells into which said vectors have beenintroduced. In one embodiment, an MP protein is prepared by using a hostcell which is cultivated in a suitable medium. The MP protein may thenbe isolated from the medium or the host cell.

Another aspect of the invention relates to a genetically modifiedmicroorganism into which an MP gene has been introduced or in which anMP gene has been modified. In one embodiment, the genome of saidmicroorganism has been modified by introducing at least one inventivenucleic acid molecule which encodes the mutated MP sequence astransgene. In another embodiment, an endogenous MP gene in the genome ofsaid microorganism has been modified, for example functionallydisrupted, by homologous recombination with a modified MP gene. In apreferred embodiment, the microorganism belongs to the genusCorynebacterium or Brevibacterium, with Corynebacterium glutamicum beingparticularly preferred. In a preferred embodiment, the microorganism isalso used for preparing a compound of interest, such as an amino acid,particularly preferably lysine.

Another preferred embodiment are host cells having more than one of thenucleic acid molecules described in Appendix A. Such host cells can beprepared in various ways known to the skilled worker. They may betransfected, for example, by vectors carrying several of the nucleicacid molecules of the invention. However, it is also possible to use avector for introducing in each case one nucleic acid molecule of theinvention into the host cell and therefore to use a plurality of vectorseither simultaneously or sequentially. Thus it is possible to constructhost cells which carry numerous, up to several hundred, nucleic acidsequences of the invention. Such an accumulation can often producesuperadditive effects on the host cell with respect to fine-chemicalproductivity.

Another aspect of the invention relates to an isolated MP protein or toa section, for example a biologically active section, thereof. In apreferred embodiment, the isolated MP protein or the section thereofregulates transcriptionally, translationally or posttranslationally oneor more metabolic pathways in C. glutamicum. In another preferredembodiment, the isolated MP protein or a section thereof is sufficientlyhomologous to an amino acid sequence of Appendix B for the protein orits section to be still capable of regulating transcriptionally,translationally or posttranslationally one or more metabolic pathways inC. glutamicum.

Moreover, the invention relates to an isolated MP-protein preparation.In preferred embodiments, the MP protein comprises an amino acidsequence of Appendix B. In a further preferred embodiment, the inventionrelates to an isolated full-length protein which is essentiallyhomologous to a complete amino acid sequence of Appendix B (which isencoded by an open reading frame in Appendix A).

The MP polypeptide or a biologically active section thereof may befunctionally linked to a non-MP polypeptide to produce a fusion protein.In preferred embodiments, this fusion protein has a different activityfrom that of the MP protein alone and, in other preferred embodiments,regulates transcriptionally, translationally or posttranslationally oneor more metabolic pathways in C. glutamicum. In particularly preferredembodiments, integration of said fusion protein into a host cellmodulates the production of a compound of interest by the cell.

Another aspect of the invention relates to a method for preparing a finechemical. The method provides for the cultivation of a cell containing avector which causes expression of an MP nucleic acid molecule of theinvention so that a fine chemical is produced. In a preferredembodiment, this method additionally comprises the step of obtaining acell containing such a vector, said cell being transfected with a vectorwhich causes expression of an MP nucleic acid. In a further preferredembodiment, said method additionally comprises the step in which thefine chemical is obtained from the culture. In a preferred embodiment,the cell belongs to the genus Corynebacterium or Brevibacterium.

Another aspect of the invention relates to methods for modulating theproduction of a molecule from a microorganism. These methods comprisecontacting the cell with a substance which modulates the MP-proteinactivity or MP nucleic-acid expression such that a cell-associatedactivity is modified in comparison with the same activity in the absenceof said substance. In a preferred embodiment, the cell is modulated withrespect to one or more regulatory systems for metabolic pathways in C.glutamicum so that this microorganism gives improved yields or animproved production rate of a fine chemical of interest. The substancewhich modulates the MP-protein activity stimulates, for example,MP-protein activity or MP nucleic-acid expression. Examples ofsubstances stimulating MP-protein activity or MP nucleic-acid expressioninclude small molecules, active MP proteins and nucleic acids whichencode MP proteins and have been introduced into the cell. Examples ofsubstances which inhibit MP activity or MP expression include smallmolecules and antisense MP nucleic acid molecules.

Another aspect of the invention relates to methods for modulating theyields of a compound of interest from a cell, comprising introducing anMP wild-type gene or MP-mutant gene into a cell, which gene eitherremains on a separate plasmid or is integrated into the genome of thehost cell. Integration into the genome may take place randomly or viahomologous recombination such that the native gene is replaced by theintegrated copy, leading to the production of the compound of interestfrom the cell to be modulated. In a preferred embodiment, said yieldsare increased. In another preferred embodiment, the chemical is a finechemical which, in a particularly preferred embodiment, is an aminoacid. In a particularly preferred embodiment, this amino acid isL-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides MP nucleic-acid and MP-protein moleculeswhich are involved in the regulation of the Corynebacterium glutamicummetabolism, including the regulation of the fine-chemical metabolism.The molecules of the invention can be used for modulating the productionof fine chemicals from microorganisms such as C. glutamicum eitherdirectly (e.g. where modulation of the activity of a regulatory proteinof the lysine metabolic pathway has a direct effect on the yield,production and/or efficiency of production of lysine from this organism)or indirectly, with the latter having nevertheless to an increase in theyield, production and/or efficiency of production of the compound ofinterest (e.g. where modulating the regulation of a nucleotidebiosynthesis protein has an effect on the production of an organic acidor fatty acid from the bacterium, possibly owing to the accompanyingregulatory modifications in the biosynthetic or degradation pathways forsaid chemicals as a reaction to the modified regulation of nucleotidebiosynthesis). The aspects of the invention are further illustratedbelow.

I. Fine Chemicals

The term “fine chemical” is known in the art and includes moleculeswhich are produced by an organism and are used in various branches ofindustry such as, for example, but not restricted to, the pharmaceuticalindustry, the agricultural industry and the cosmetics industry. Thesecompounds comprise organic acids such as tartaric acid, itaconic acidand diaminopimelic acid, both proteinogenic and nonproteinogenic aminoacids, purine and pyrimidine bases, nucleosides and nucleotides (asdescribed, for example, in Kuninaka, A. (1996) Nucleotides and relatedcompounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., editorsVCH: Weinheim and the references therein), lipids, saturated andunsaturated fatty acids (for example arachidonic acid), diols (e.g.propanediol and butanediol), carbohydrates (e.g. hyaluronic acid andtrehalose), aromatic compounds (e.g. aromatic amines, vanillin andindigo), vitamins and cofactors (as described in Ullmann's Encyclopediaof Industrial Chemistry, Vol. A27, “Vitamins”, pp. 443-613 (1996) VCH:weinheim and the references therein; and Ong, A. S., Niki, E. andPacker, L. (1995) “Nutrition, Lipids, Health and Disease” Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research—Asia, held Sep. 1-3,1994, in Penang, Malaysia, AOCS Press (1995)), enzymes and all otherchemicals described by Gutcho (1983) in Chemicals by Fermentation, NoyesData Corporation, ISBN: 0818805086 and the references indicated therein.The metabolism and the uses of particular fine chemicals are furtherillustrated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the fundamental structural units of all proteinsand are thus essential for normal functions of the cell. The term “aminoacid” is known in the art. Proteinogenic amino acids, of which there are20 types, serve as structural units for proteins, in which they arelinked together by peptide bonds, whereas the nonproteinogenic aminoacids (hundreds of which are known) usually do not occur in proteins(see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97VCH: Weinheim (1985)). Amino acids can exist in the D or Lconfiguration, although L-amino acids are usually the only type found innaturally occurring proteins. Biosynthetic and degradation pathways ofeach of the 20 proteinogenic amino acids are well characterized both inprokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3rd edition, pp. 578-590 (1988)). The “essential” aminoacids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan and valine), so called because,owing to the complexity of their biosyntheses, they must be taken inwith the diet, are converted by simple biosynthetic pathways into theother 11 “nonessential” amino acids (alanine, arginine, asparagine,aspartate, cysteine, glutamate, glutamine, glycine, proline, serine andtyrosine). Higher animals are able to synthesize some of these aminoacids but the essential amino acids must be taken in with the food inorder that normal protein synthesis takes place.

Apart from their function in protein biosynthesis, these amino acids areinteresting chemicals as such, and it has been found that many havevarious applications in the food, animal feed, chemicals, cosmetics,agricultural and pharmaceutical industries. Lysine is an important aminoacid not only for human nutrition but also for monogastric livestocksuch as poultry and pigs. Glutamate is most frequently used as flavoradditive (monosodium glutamate, MSG) and elsewhere in the food industry,as are aspartate, phenylalanine, glycine and cysteine. Glycine,L-methionine and tryptophan are all used in the pharmaceutical industry.Glutamine, valine, leucine, isoleucine, histidine, arginine, proline,serine and alanine are used in the pharmaceutical industry and thecosmetics industry. Threonine, tryptophan and D/L-methionine are widelyused animal feed additives (Leuchtenberger, W. (1996) Aminoacids—technical production and use, pp. 466-502 in Rehm et al.,(editors) Biotechnology Vol. 6, Chapter 14a, VCH: Weinheim). It has beenfound that these amino acids are additionally suitable as precursors forsynthesizing synthetic amino acids and proteins, such asN-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophanand other substances described in Ullmann's Encyclopedia of IndustrialChemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms able toproduce them, for example bacteria, has been well characterized (for areview of bacterial amino acid biosynthesis and its regulation, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by reductive amination of α-ketoglutarate, an intermediateproduct in the citric acid cycle. Glutamine, proline and arginine areeach generated successively from glutamate. The biosynthesis of serinetakes place in a three-step process and starts with 3-phosphoglycerate(an intermediate product of glycolysis), and affords this amino acidafter oxidation, transamination and hydrolysis steps. Cysteine andglycine are each produced from serine, specifically the former bycondensation of homocysteine with serine, and the latter by transfer ofthe side-chain β-carbon atom to tetrahydrofolate in a reaction catalyzedby serine transhydroxymethylase. Phenylalanine and tyrosine aresynthesized from the precursors of the glycolysis and pentose phosphatepathway, and erythrose 4-phosphate and phosphoenolpyruvate in a 9-stepbiosynthetic pathway which diverges only in the last two steps after thesynthesis of prephenate. Tryptophan is likewise produced from these twostarting molecules but it is synthesized by an 11-step pathway. Tyrosinecan also be prepared from phenylalanine in a reaction catalyzed byphenylalanine hydroxylase. Alanine, valine and leucine are eachbiosynthetic products derived from pyruvate, the final product ofglycolysis. Aspartate is formed from oxalacetate, an intermediateproduct of the citrate cycle. Asparagine, methionine, threonine andlysine are each produced by the conversion of aspartate. Isoleucine isformed from threonine. Histidine is formed from 5-phosphoribosyl1-pyrophosphate, an activated sugar, in a complex 9-step pathway.

Amounts of amino acids exceeding those required for protein biosynthesisby the cell cannot be stored and are instead broken down so thatintermediate products are provided for the principal metabolic pathwaysin the cell (for a review, see Stryer, L., Biochemistry, 3rd edition,Chapter 21 “Amino Acid Degradation and the Urea Cycle”; pp. 495-516(1988)). Although the cell is able to convert unwanted amino acids intothe useful intermediate products of metabolism, production of aminoacids is costly in terms of energy, the precursor molecules and theenzymes necessary for their synthesis. It is therefore not surprisingthat amino acid biosynthesis is regulated by feedback inhibition,whereby the presence of a particular amino acid slows down or completelystops its own production (for a review of the feedback mechanism inamino acid biosynthetic pathways, see Stryer, L., Biochemistry, 3rdedition, Chapter 24, “Biosynthesis of Amino Acids and Heme”, pp. 575-600(1988)). The output of a particular amino acid is therefore restrictedby the amount of this amino acid in the cell.

B. Vitamins, Cofactors and Nutraceutical Metabolism, and Uses

Vitamins, cofactors and nutraceuticals comprise another group ofmolecules. Higher animals have lost the ability to synthesize them andtherefore have to take them in, although they are easily synthesized byother organisms such as bacteria. These molecules are either bioactivemolecules per se or precursors of bioactive substances which serve aselectron carriers or intermediate products in a number of metabolicpathways. Besides their nutritional value, these compounds also have asignificant industrial value as colorants, antioxidants and catalysts orother processing auxiliaries. (For a review of the structure, activityand industrial applications of these compounds, see, for example,Ullmann's Encyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27,pp. 443-613, VCH: Weinheim, 1996). The term “vitamin” is known in theart and comprises nutrients which are required for normal functioning ofan organism but cannot be synthesized by this organism itself. The groupof vitamins may include cofactors and nutraceutical compounds. The term“cofactor” comprises nonproteinaceous compounds necessary for theappearance of normal enzymic activity. These compounds may be organic orinorganic; the cofactor molecules of the invention are referablyorganic. The term “nutraceutical” comprises food additives which arehealth-promoting in plants and animals, especially humans. Examples ofsuch molecules are vitamins, antioxidants and likewise certain lipids(e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms able to produce them,such as bacteria, has been comprehensively characterized (Ullmann'sEncyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27, pp. 443-613,VCH: Weinheim, 1996, Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. and Packer, L. (1995) “Nutrition, Lipids, Health and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia and the Society for Free Radical Research-Asia,held on Sep. 1-3, 1994, in Penang, Malaysia, AOCS Press, Champaign, Ill.X, 374 S).

Thiamine (vitamin B₁) is formed by chemical coupling of pyrimidine andthiazole units. Riboflavin (vitamin B₂) is synthesized from guanosine5′-triphosphate (GTP) and ribose 5′-phosphate. Riboflavin in turn isemployed for the synthesis of flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD). The family of compounds together referred toas “vitamin B6” (for example pyridoxine, pyridoxamine, pyridoxal5′-phosphate and the commercially used pyridoxine hydrochloride), areall derivatives of the common structural unit5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,R-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beprepared either by chemical synthesis or by fermentation. The last stepsin pantothenate biosynthesis consist of ATP-driven condensation ofβ-alanine and pantoic acid. The enzymes responsible for the biosyntheticsteps for the conversion into pantoic acid and into β-alanine and forthe condensation to pantothenic acid are known. The metabolically activeform of pantothenate is coenzyme A whose biosynthesis takes place by 5enzymatic steps. Pantothenate, pyridoxal 5′-phosphate, cysteine and ATPare the precursors of coenzyme A. These enzymes catalyze not only theformation of pantothenate but also the production of (R)-pantoic acid,(R)-pantolactone, (R)-panthenol (provitamin B₅), pantetheine (and itsderivatives) and coenzyme A.

The biosynthesis of biotin from the precursor molecule pimeloyl-CoA inmicroorganisms has been investigated in detail, and several of the genesinvolved have been identified. It has emerged that many of thecorresponding proteins are involved in the Fe cluster synthesis andbelong to the class of nifS proteins. Liponic acid is derived fromoctanonoic acid and serves as coenzyme in energy metabolism where it isa constituent of the pyruvate dehydrogenase complex and of theα-ketoglutarate dehydrogenase complex. Folates are a group of substancesall derived from folic acid which in turn is derived from L-glutamicacid, p-aminobenzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives starting from the metabolic intermediateproducts of the biotransformation of guanosine 5′-triphosphate (GTP),L-glutamic acid and p-aminobenzoic acid has been investigated in detailin certain microorganisms.

Corrinoids (such as the cobalamines and, in particular, vitamin B₁₂) andthe porphyrins belong to a group of chemicals distinguished by atetrapyrrole ring system. The biosynthesis of vitamin B₁₂ is so complexthat it has not yet been completely characterized, but many of theenzymes and substrates involved are now known. Nicotinic acid(nicotinate) and nicotinamide are pyridine derivatives which are alsoreferred to as “niacin”. Niacin is the precursor of the importantcoenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamideadenine dinucleotide phosphate) and their reduced forms.

Production of these compounds on the industrial scale is mostly based oncell-free chemical syntheses, although some of these chemicals havelikewise been produced by large-scale cultivation of microorganisms,such as riboflavin, vitamin B₆, pantothenate and biotin. Only vitaminB₁₂ is, because of the complexity of its synthesis, produced only byfermentation. In vitro processes require a considerable expenditure ofmaterials and time and frequently high costs.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Genes for purine and pyrimidine metabolism and their correspondingproteins are important aims for the therapy of oncoses and viralinfections. The term “purine” or “pyrimidine” comprisesnitrogen-containing bases which form part of nucleic acids, coenzymesand nucleotides. The term “nucleotide” encompasses the fundamentalstructural units of nucleic acid molecules, which comprise anitrogen-containing base, a pentose sugar (the sugar is ribose in thecase of RNA and the sugar is D-deoxyribose in the case of DNA) andphosphoric acid. The term “nucleoside” comprises molecules which serveas precursors of nucleotides but have, in contrast to the nucleotides,no phosphoric acid unit. It is possible to inhibit RNA and DNA synthesisby inhibiting the biosynthesis of these molecules or their mobilizationto form nucleic acid molecules; targeted inhibition of this activity incancerogenic cells allows the ability of tumor cells to divide andreplicate to be inhibited.

There are also nucleotides which do not form nucleic acid molecules butserve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, the purine and/or pyrimidine metabolism beinginfluenced (for example Christopherson, R. I. and Lyons, S. D. (1990)“Potent inhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents”, Med. Res. Reviews 10: 505-548). Investigationsof enzymes involved in purine and pyrimidine metabolism haveconcentrated on the development of novel medicaments which can be used,for example, as immunosuppressants or antiproliferative agents (Smith,J. L. “Enzymes in Nucleotide Synthesis” Curr. Opin. Struct. Biol. 5(1995) 752-757; Biochem. Soc. Transact. 23 (1995) 877-902). However,purine and pyrimidine bases, nucleosides and nucleotides also have otherpossible uses: as intermediate products in the biosynthesis of variousfine chemicals (e.g. thiamine, S-adenosylmethionine, folates orriboflavin), as energy carriers for the cell (for example ATP or GTP)and for chemicals themselves, are ordinarily used as flavor enhancers(for example IMP or GMP) or for many medical applications (see, forexample, Kuninaka, A., (1996) “Nucleotides and Related Compounds inBiotechnology” Vol. 6, Rehm et al., editors VCH: Weinheim, pp. 561-612).Enzymes involved in purine, pyrimidine, nucleoside or nucleotidemetabolism are also increasingly serving as targets against whichchemicals are being developed for crop protection, including fungicides,herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized(for reviews, see, for example, Zalkin, H. and Dixon, J. E. (1992) “Denovo purine nucleotide biosynthesis” in Progress in Nucleic AcidsResearch and Molecular biology, Vol. 42, Academic Press, pp. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”; Chapter 8 in :Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley, New York). Purine metabolism, the object of intensive research,is essential for normal functioning of the cell. Disordered purinemetabolism in higher animals may cause severe illnesses, for examplegout. Purine nucleotides are synthesized from ribose 5-phosphate by anumber of steps via the intermediate compound inosine 5′-phosphate(IMP), leading to the production of guanosine 5′-monophosphate (GMP) oradenosine 5′-monophosphate (AMP), from which the triphosphate forms usedas nucleotides can easily be prepared. These compounds are also used asenergy stores, so that breakdown thereof provides energy for manydifferent biochemical processes in the cell. Pyrimidine biosynthesistakes place via formation of uridine 5′-monophosphate (UMP) from ribose5-phosphate. UMP in turn is converted into cytidine 5′-triphosphate(CTP). The deoxy forms of all nucleotides are prepared in a one-stepreduction reaction from the diphosphate ribose form of the nucleotide togive the diphosphate deoxyribose form of the nucleotide. Afterphosphorylation, these molecules can take part in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules linked together by α,α-1,1linkage. It is ordinarily used in the food industry as sweetener, asadditive for dried or frozen foods and in beverages. However, it is alsoused in the pharmaceutical industry or in the cosmetics industry andbiotechnology industry (see, for example, Nishimoto et al., (1998) U.S.Pat. No. 5,759,610; Singer, M. A. and Lindquist, S. Trends Biotech. 16(1998) 460-467; Paiva, C. L. A. and Panek, A. D. Biotech Ann. Rev. 2(1996) 293-314; and Shiosaka, M. J. Japan 172 (1997) 97-102). Trehaloseis produced by enzymes of many microorganisms and is naturally releasedinto the surrounding medium from which it can be isolated by methodsknown in the art.

II. Mechanisms of Metabolic Regulation

All living cells have complex catabolic and anabolic capabilities withmany metabolic pathways linked to one another. In order to maintain anequilibrium between the various parts of this extremely complexmetabolic network, the cell employs a finely tuned regulatory network.By regulating the enzyme synthesis and enzyme activity, eitherindependently or simultaneously, the cell can regulate the activity ofcompletely different metabolic pathways so as to meet the cell'schanging needs.

The induction or repression of enzyme synthesis may take place either atthe transcriptional or the translational level or at both levels (for areview, see Lewin, B. (1990) Genes IV, Part 3: “Controlling prokaryoticgenes by transcription”, Oxford University Press, Oxford, pp. 213-301,and the references therein, and Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons). Allof these known regulatory processes are mediated by additional geneswhich themselves react to various external influences (e.g. temperature,nutrient supply or light). Examples of protein factors involved in thistype of regulation include the transcription factors. These are proteinswhich bind to the DNA, thereby causing the expression of a gene eitherto increase (positive regulation as in the case of the E. coli araoperon) or to decrease (negative regulation as in the case of the E.coli lac operon). These expression-modulating transcription factors maythemselves be subject to regulation. Their activity may be regulated,for example, by low molecular weight compounds binding to theDNA-binding protein, whereby binding of said proteins to the appropriatebinding site on the DNA is stimulated (as in the case of arabinose forthe ara operon) or inhibited (as in the case of lactose for the lacoperon) (see, for example, Helmann, J. D. and Chamberlin, M. J. (1988)“Structure and function of bacterial sigma factors” Ann. Rev. Biochem.57: 839-872; Adhya, S. (1995) “The lac and gal operons today” and Boos,W. et al., “The maltose system”, both in Regulation of Gene Expressionin Escherichia coli (Lin, E. C. C. and Lynch, A. S. editors) Chapman &Hall: New York, pp. 181-200 and 201-229; and Moran, C. P. (1993) “RNApolymerase and transcription factors” in: Bacillus subtilis and otherGram-positive bacteria, Sonenshein, A. L. editors ASM: Washington D.C.pp. 653-667).

Protein synthesis is regulated not only at the transcriptional level butoften also at the translational level. This regulation can be carriedout via many mechanisms, including modification of the ability of theribosome to bind to one or more mRNAs, the binding of the ribosome tomRNA, maintaining or removing of the mRNA secondary structure, usingcommon or less common codons for a particular gene, the degree ofabundance of one or more tRNAs and specific regulatory mechanisms suchas attenuation (see Vellanoweth, R. I. (1993) Translation and itsregulation in Bacillus subtilis and other Gram-positive bacteria,Sonenshein, A. L. et al. editors ASM: Washington, D.C., pp. 699-711 andreferences therein.

The transcriptional and translational regulation may be directed towarda single protein (sequential regulation) or simultaneously toward aplurality of proteins in various metabolic pathways (coordinatedregulation). Genes whose expression is regulated in a coordinatedfashion are often located in the genome in close proximity in an operonor regulon. This up or down regulation of gene transcription and genetranslation is controlled by the cellular or extracellular amounts ofvarious factors such as substrates (precursors and intermediates whichare used in one or more metabolic pathways), catabolites (moleculesproduced by biochemical pathways which are connected with energyproduction from the degradation of complex organic molecules such assugars) and end products (molecules which are obtained at the end of ametabolic pathway). The expression of genes which encode enzymesrequired for the activity of a particular metabolic pathway is inducedby large amounts of substrate molecules for said metabolic pathway.Correspondingly, this gene expression is repressed by the presence oflarge intracellular amounts of the end product of the pathway (Snyder,L. and Champness, W. (1977) The Molecular Biology of Bacteria ASM:Washington). The gene expression may likewise be regulated by otherexternal and internal factors such as environmental conditions (e.g.heat, oxidative stress or hunger). These global environmental changescause changes in the expression of specialized modulating genes whichtrigger gene expression directly or indirectly (via additional genes orproteins) by binding to DNA and thereby induce or repress transcription(see, for example, Lin, E. C. C. and Lynch, A. S. editors (1995)Regulation of Gene Expression in Escherichia coli, Chapman & Hall: NewYork).

Another mechanism by which the cellular metabolism can be regulatedtakes place at the protein level. This regulation is carried out eithervia the activities of other enzymes or via binding of low molecularweight components which prevent or enable normal function of theprotein. Examples of protein regulation by binding of low molecularweight compounds include the binding of GTP or NAD. The binding of lowmolecular weight chemicals is usually reversible, for example in thecase of GTP-binding proteins. These proteins occur in two states (withbound GTP or GDP), with one state being the active form of the proteinand the other one the inactive form.

The protein activity is regulated by the action of other enzymes usuallyvia covalent modification of the protein (i.e. phosphorylation of aminoacid residues such as histidine or aspartate or methylation). Thiscovalent modification is usually reversible and this is effected by anenzyme having the opposite activity. An example for this is the oppositeactivity of kinases and phosphorylases in protein phosphorylation:protein kinases phosphorylate specific residues on a target protein(e.g. serine or threonine), whereas protein phosphorylases remove thephosphate groups from said proteins. Enzymes modulating the activity ofother proteins are usually modulated themselves by external stimuli.These stimuli are mediated by proteins acting as sensors. A well-knownmechanism by which these sensor proteins mediate said external signalsis dimerization, but other mechanisms are also known (see, for example,Msadek, T. et al. (1993) “Two-component Regulatory Systems” in: Bacillussubtilis and Other Gram-Positive Bacteria, Sonenshein, A. L. et al.,editors, ASM: Washington, pp. 729-745 and references therein).

A detailed understanding of the regulatory networks which control thecellular metabolism in microorganisms is crucial for the production ofchemicals in high yields by fermentation. Control systems fordownregulating the metabolic pathways may be removed or reduced in orderto improve the synthesis of chemicals of interest and, correspondingly,those for upregulation of the metabolic pathway of a product of interestmay be constitutively activated or optimized with respect to theactivity (as shown in Hirose, Y. and Okada, H. (1979) “MicrobialProduction of Amino Acids”, in: Peppler, H. J. and Perlman, D. (editors)Microbial Technology 2nd edition, Vol. 1, Chapter 7, Academic Press, NewYork).

III. Elements and Methods of the Invention

The present invention is based, at least partially, on the detection ofnew molecules which are referred to herein as MP nucleic-acid andMP-protein molecules and which regulate one or more metabolic pathwaysin C. glutamicum by transcriptional, translational or posttranslationalmeasures. In one embodiment, the MP molecules regulatetranscriptionally, translationally or posttranslationally a metabolicpathway in C. glutamicum. In a preferred embodiment, the activity of theinventive MP molecules for regulating one or more metabolic pathways inC. glutamicum has an effect on the production of a fine chemical ofinterest by said organism. In a particularly preferred embodiment, theMP molecules of the invention have a modulated activity so that the C.glutamicum metabolic pathways regulated by the MP proteins of theinvention are modulated with respect to their efficiency or theirthroughput and this modulates either directly or indirectly the yield,production and/or efficiency of production of a fine chemical ofinterest by C. glutamicum.

The term “MP protein” or “MP polypeptide” comprises proteins whichregulate transcriptionally, translationally or posttranslationally ametabolic pathway in C. glutamicum. Examples of MP proteins comprisethose which are encoded by the MP genes listed in Table 1 and AppendixA. The terms “MP gene” and “MP nucleic acid sequence” comprise nucleicacid sequences encoding an MP protein which comprises a coding regionand corresponding untranslated 5′ and 3′ sequence regions. Examples ofMP genes are listed in Table 1. The terms “production” and“productivity” are known in the art and include the concentration of thefermentation product (for example the fine chemical of interest, whichis produced within a predetermined time interval and a predeterminedfermentation volume (e.g. kg of product per h per l). The term“efficiency of production” comprises the time required for attaining aparticular production quantity (for example, the time required by thecell for reaching a particular throughput rate of a fine chemical). Theterm “yield” or “product/carbon yield” is known in the art and comprisesthe efficiency of converting the carbon source into the product (i.e.the fine chemical). This is, for example, usually expressed as kg ofproduct per kg of carbon source. Increasing the yield or production ofthe compound increases the amount of the molecules obtained or of thesuitable obtained molecules of this compound in a particular culturevolume over a predetermined period. The terms “biosynthesis” and“biosynthetic pathway” are known in the art and comprise the synthesisof a compound, preferably an organic compound, from intermediates by acell, for example in a multistep process or highly regulated process.The terms “degradation” and “degradation pathway” are known in the artand comprise cleavage of a compound, preferably an organic compound,into degradation products (in more general terms: smaller or lesscomplex molecules) by a cell, for example in a multistep process orhighly regulated process. The term “metabolism” is known in the art andcomprises the entirety of biochemical reactions which take place in anorganism. The metabolism of a particular compound (e.g. the metabolismof an amino acid such as glycine) then comprises all biosynthetic,modification and degradation pathways of this compound in the cell. Theterm “regulation” is known in the art and comprises the activity of aprotein for controlling the activity of another protein. The term“transcriptional regulation” is known in the art and comprises theactivity of a protein for inhibiting or activating the conversion of aDNA encoding a target protein into mRNA. The term “translationalregulation” is known in the art and comprises the activity of a proteinfor inhibiting or activating conversion of an mRNA encoding a targetprotein into a protein molecule. The term “posttranslational regulation”is known in the art and comprises the activity of a protein forinhibiting or improving the activity of a target protein by covalentlymodifying the target protein (e.g. by methylation, glycosylation orphosphorylation).

In another embodiment the MP molecules of the invention are capable ofmodulating the production of a molecule of interest, such as a finechemical, in a microorganism such as C. glutamicum. With the aid of generecombination techniques it is possible to manipulate one or moreinventive regulatory proteins for metabolic pathways such that theirfunction is modulated. It is possible, for example, to improve abiosynthesis enzyme with respect to efficiency or to destroy itsallosteric control region so that feedback inhibition of the productionof the compound is prevented. Accordingly, a degradation enzyme can bedeleted or be modified by substitution, deletion or addition such thatits degradation activity for the compound of interest is reduced,without impairing cell viability. In any case, it is possible toincrease the overall yield or production rate of any of said finechemicals of interest.

It is also possible that these modifications in the protein andnucleotide molecules of the invention can improve the production of finechemicals indirectly. The regulatory mechanisms of the metabolicpathways in the cell are inevitably linked and activation of onemetabolic pathway can cause repression or activation of anothermetabolic pathway in an accompanying manner. Modulating the activity ofone or more proteins of the invention can influence the production orthe efficiency of the activity of other fine-chemical biosynthetic ordegradation pathways. Reducing the ability of an MP protein to repressthe transcription of a gene which encodes a particular protein in aminoacid biosynthesis makes it possible to simultaneously derepress otheramino acid biosynthetic pathways, since these metabolic pathways arelinked to one another. By modifying the MP proteins of the invention itis possible to decouple to a certain degree cell growth and celldivision from their extracellular environments; by influencing an MPprotein which usually represses the biosynthesis of a nucleotide whenthe extracellular conditions for growth and cell division are suboptimalsuch that it now lacks this function, it is possible to enable growtheven if the extracellular conditions are poor. This is of particularimportance for large-scale fermentative cultivation for which theculture conditions with respect to temperature, nutrient supply oraeration are often suboptimal but can still promote growth and celldivision, after the cellular regulatory systems for said factors havebeen eliminated.

A suitable starting point for preparing the nucleic acid sequences ofthe invention is the genome of a Corynebacterium glutamicum strain whichcan be obtained from the American Type Culture Collection under the nameATCC 13032.

The nucleic acid sequences of the invention can be prepared from thesenucleic acid sequences via the modifications denoted in Table 1, usingconventional methods.

The MP protein of the invention or a biologically active section orfragments thereof can regulate transcriptionally, translationally orposttranslationally a metabolic pathway in C. glutamicum or can have oneor more of the activities listed in Table 1.

The following subsections describe various aspects of the invention inmore detail:

A. Isolated Nucleic Acid Molecules

One aspect of the invention relates to isolated nucleic acid moleculeswhich encode MP polypeptides or biologically active sections thereof andto nucleic acid fragments which are sufficient for use as hybridizationprobes or primers for identifying or amplifying MP-encoding nucleicacids (e.g. MP DNA). The term “nucleic acid molecule”, as used herein,is intended to comprise DNA molecules (e.g. cDNA or genomic DNA) and RNAmolecules (e.g. mRNA) and also DNA or RNA analogs which are generated bymeans of nucleotide analogs. Moreover, this term comprises theuntranslated sequence located at the 3′ and 5′ ends of the coding generegion: at least about 100 nucleotides of the sequence upstream of the5′ end of the coding region and at least about 20 nucleotides of thesequence downstream of the 3′ end of the coding gene region. The nucleicacid molecule may be single-stranded or double-stranded but ispreferably a double-stranded DNA. An “isolated” nucleic acid molecule isremoved from other nucleic acid molecules which are present in thenatural source of the nucleic acid. An “isolated” nucleic acidpreferably does not have any sequences which flank the nucleic acidnaturally in the genomic DNA of the organism from which the nucleic acidoriginates (for example sequences located at the 5′ or 3′ end of thenucleic acid). In various embodiments, the isolated MP nucleic acidmolecule may, for example, less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of the nucleotide sequences which naturally flank thenucleic acid molecule in the genomic DNA of the cell from which thenucleic acid originates (e.g. a C. glutamicum cell). In addition tothis, an “isolated” nucleic acid molecule, such as a cDNA molecule, maybe essentially free of another cellular material or culture medium, ifprepared by recombinant techniques, or free of chemical precursors orother chemicals, if it is chemically synthesized.

A nucleic acid molecule of the invention, for example a nucleic acidmolecule having a nucleotide sequence of Appendix A or a sectionthereof, may be prepared by means of molecular biological standardtechniques and the sequence information provided here. For example, a C.glutamicum MP cDNA may be isolated from a C. glutamicum bank by using acomplete sequence from Appendix A or a section thereof as hybridizationprobe and by using standard hybridization techniques (as described, forexample, in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).Moreover, a nucleic acid molecule comprising a complete sequence fromAppendix A or a section thereof can be isolated via polymerase chainreaction, using the oligonucleotide primers produced on the basis ofsaid sequence (for example, it is possible to isolate a nucleic acidmolecule comprising a complete sequence from Appendix A or a sectionthereof via polymerase chain reaction by using oligonucleotide primerswhich have been produced on the basis of this same sequence fromAppendix A). For example, mRNA can be isolated from normal endothelialcells (for example via the guanidinium-thiocyanate extraction method ofChirgwin et al. (1979) Biochemistry 18: 5294-5299), and the cDNA can beprepared by means of reverse transcriptase (e.g. Moloney-MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md., or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for amplification viapolymerase chain reaction can be produced on the basis of any of thenucleotide sequences shown in Appendix A. A nucleic acid of theinvention may be amplified by means of cDNA or, alternatively, genomicDNA as template and suitable oligonucleotide primers according to PCRstandard amplification techniques. The nucleic acid amplified in thisway may be cloned into a suitable vector and characterized by DNAsequence analysis. Oligonucleotides corresponding to an MP nucleotidesequence may be prepared by standard syntheses using, for example, anautomatic DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises any of the nucleotide sequences listed in AppendixA.

In a further preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule complementary to any ofthe nucleotide sequences shown in Appendix A or a section thereof, saidnucleic acid molecule being sufficiently complementary to any of thenucleotide sequences shown in Appendix A for it to hybridize with any ofthe sequences indicated in Appendix A, resulting in a stable duplex.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or a section thereof comprising an amino acid sequence which issufficiently homologous to an amino acid sequence from Appendix B forthe protein or a section thereof to be still capable of regulating ametabolic pathway in C. glutamicum transcriptionally, translationally orposttranslationally. The term “sufficiently homologous”, as used herein,relates to proteins or sections thereof whose amino acid sequences havea minimum number of identical or equivalent amino acid residues (forexample an amino acid residue having a side chain similar to that of anamino acid residue in any of the sequences from Appendix B) compared toan amino acid sequence from Appendix B so that the protein or a sectionthereof can transcriptionally, translationally or posttranslationallyregulate a metabolic pathway in C. glutamicum. Protein components ofthese metabolic pathways, as described herein, may regulate thebiosynthesis or degradation of one or more fine chemicals. Examples ofthese activities are likewise described herein. Thus the “function of anMP protein” relates to the overall regulation of one or more metabolicfine-chemical pathways. Table 1 lists examples of MP protein activities.

Sections of proteins encoded by the MP nucleic acid molecules of theinvention are preferably biologically active sections of any of the MPproteins. The term “biologically active section of an MP protein”, asused herein, is intended to comprise a section, for example a domain ora motive, of an MP protein, which can transcriptionally, translationallyor posttranslationally regulate a metabolic pathway in C. glutamicum orhas an activity indicated in Table 1. In order to determine whether anMP protein or a biologically active section thereof can regulate ametabolic pathway in C. glutamicum transcriptionally, translationally orposttranslationally, an enzyme activity assay may be carried out. Theseassay methods, as described in detail in Example 8 of the examples, arefamiliar to the skilled worker.

In addition to naturally occuring MP-sequence variants which may existin the population, the skilled worker also understands that changes canbe introduced into a nucleotide sequence from Appendix A via mutation,leading to a change in the amino acid sequence of the encoded MPprotein, without impairing the functionality of the MP protein. Thus itis possible, for example, to prepare in a sequence from Appendix Anucleotide substitutions which lead to amino acid substitutions at“nonessential” amino acid residues. A “nonessential” amino acid residuein a wild-type sequence of any of the MP proteins (Appendix B) can bemodified without modifying the activity of said MP protein, whereas an“essential” amino acid residue is required for MP-protein activity.However, other amino acid residues (for example nonconserved or merelysemiconserved amino acid residues in the domain with MP activity) may benonessential for activity and can therefore probably be modified withoutmodifying the MP activity.

An isolated nucleic acid molecule encoding an MP protein which ishomologous to a protein sequence from Appendix B may be generated byintroducing one or more nucleotide substitutions, additions or deletionsinto a nucleotide sequence from Appendix A so that one or more aminoacid substitutions, additions or deletions are introduced into theencoded protein. The mutations may be introduced into any of thesequences from Appendix A by standard techniques such as site-directedmutagenesis and PCR-mediated mutagenesis. Preference is given tointroducing conservative amino acid substitutions at one or more of thepredicted nonessential amino acid residues. A “conservative amino acidsubstitution” replaces the amino acid residue by an amino acid residuewith a similar side chain. Families of amino acid residues with similarside chains have been defined in the art. These families comprise aminoacids with basic side chains (e.g. lysine, arginine, histidine), acidicside chains (e.g. aspartic acid, glutamic acid), uncharged polar sidechains (e.g. glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g. threonine, valine, isoleucine) andaromatic side chains (e.g. tyrosine, phenylalanine, tryptophan,histidine). A predicted nonessential amino acid residue in an MP proteinis thus preferably replaced by another amino acid residue of the sameside-chain family. In another embodiment, the mutations mayalternatively be introduced randomly over the entire or over part of theMP-encoding sequence, for example by saturation mutagenesis, and theresulting mutants may be tested for the MP activity described here inorder to identify mutants maintaining MP activity. After mutagenesis ofany of the sequences from Appendix A, the encoded protein may beexpressed recombinantly, and the activity of said protein may bedetermined, for example, using the assays described herein (see Example8 of the examples).

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to vectors, preferablyexpression vectors, containing a nucleic acid which encodes an MPprotein (or a section thereof). The term “vector”, as used herein,relates to a nucleic acid molecule capable of transporting anothernucleic acid to which it is bound. One type of vector is a “plasmid”which term means a circular double-stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, in the case of which additional DNA segments can beligated into the viral genome. Certain vectors are capable ofreplicating autonomously in a host cell into which they have beenintroduced (for example bacterial vectors with bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.nonepisomal mammalian vectors) are integrated into the genome of a hostcell when introduced into said host cell and thereby replicated togetherwith the host genome. Moreover, particular vectors are capable ofcontrolling the expression of genes to which they are functionallylinked. These vectors are referred to as “expression vectors”. Normally,expression vectors used in DNA recombination techniques are in the formof plasmids. In the present description, “plasmid” and “vector” may beused interchangeably, since the plasmid is the most commonly used typeof vector. The invention is intended to comprise said other types ofexpression vectors such as viral vectors (for example,replication-deficient retroviruses, adenoviruses and adenovirus-relatedviruses), which exert similar functions.

The recombinant expression vector of the invention comprises a nucleicacid of the invention in a form which is suitable for expressing saidnucleic acid in a host cell, meaning that the recombinant expressionvectors comprise one or more regulatory sequences which are selected onthe basis of the host cells to be used for expression and which arefunctionally linked to the nucleic acid sequence to be expressed. In arecombinant expression vector, the term “functionally linked” means thatthe nucleotide sequence of interest is bound to the regulatorysequence(s) such that expression of said nucleotide sequence is possible(for example in an in vitro transcription/translation system or in anyhost cell, if the vector has been introduced into said host cell). Theterm “regulatory sequence” is intended to comprise promoters, enhancersand other expression control elements (e.g. polyadenylation signals).These regulatory sequences are described, for example, in Goeddel: GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences comprise those which controlconstitutive expression of a nucleotide sequence in many types of hostcells and those which control direct expression of the nucleotidesequence only in particular host cells. The skilled worker understandsthat designing an expression vector may depend on factors such as thechoice of host cell to be transformed, the extent of expression of theprotein of interest, etc. The expression vectors of the invention may beintroduced into the host cells in order to prepare proteins or peptides,including fusion proteins or fusion peptides, which are encoded by thenucleic acids as described herein (e.g. MP proteins, mutated forms of MPproteins, fusion proteins, etc.).

The recombinant expression vectors of the invention may be designed forexpressing MP proteins in prokaryotic or eukaryotic cells. For example,MP genes may be expressed in bacterial cells such as C. glutamicum,insect cells (using Baculovirus expression vectors), yeast cells andother fungal cells (see Romanos, M. A. et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A.M. J. J. et al. (1991) “Heterologous gene expression in filamentousfungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, editors, pp. 396-428: Academic Press: San Diego; and van denHondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems andvector development for filamentous fungi” in: Applied Molecular Geneticsof Fungi, Peberdy, J. F. et al., editors, pp. 1-28, Cambridge UniversityPress: Cambridge), algal and multicellular plant cells (see Schmidt, R.and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.: 583-586) or mammalian cells.Suitable host cells are further discussed in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector may betranscribed and translated in vitro, for example by using T7 promoterregulatory sequences and T7 polymerase.

Proteins are expressed in prokaryotes mainly by using vectors containingconstitutive or inducible promoters which control the expression offusion or nonfusion proteins. Fusion vectors contribute a number ofamino acids to a protein encoded therein, usually at the amino terminusof the recombinant protein. These fusion vectors usually have threetasks: 1) enhancing the expression of recombinant protein; 2) increasingthe solubility of the recombinant protein; and 3) supporting thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often a proteolytic cleavage site is introducedinto fusion expression vectors at the junction of fusion unit andrecombinant protein so that the recombinant protein can be separatedfrom the fusion unit after purifying the fusion protein. These enzymesand their corresponding recognition sequences comprise factor Xa,thrombin and enterokinase.

Common fusion expression vectors comprise PGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), in which glutathione S-transferase (GST), maltose E-bindingprotein and protein A, respectively, are fused to the recombinant targetprotein. In one embodiment, the coding sequence of the MP protein iscloned into a pGEX expression vector such that a vector is generated,which encodes a fusion protein comprising, from N terminus to Cterminus, GST—thrombin cleavage site—protein X. The fusion protein maybe purified via affinity chromatography by means of aglutathione-agarose resin. The recombinant MP protein which is not fusedto GST may be obtained by cleaving the fusion protein with thrombin.

Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d(Studier et al. Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). The target geneexpression from the pTrc vector is based on transcription from a hybridtrp-lac fusion promoter by host RNA polymerase. The target geneexpression from the pET11d vector is based on transcription from aT7-gn10-lac fusion promoter, which is mediated by a coexpressed viralRNA polymerase (T7 gn1). This viral polymerase is provided by the BL 21(DE3) or HMS174 (DE3) host strain by a resident λ prophage which harborsa T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

One strategy for maximizing expression of the recombinant protein is toexpress said protein in a host bacterium whose ability toproteolytically cleave said recombinant protein is disrupted (Gottesman,S. Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to modifythe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector such that the individual codons for each amino acidare those which are preferably used in a bacterium selected forexpression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res.20: 2111-2118). This modification of the nucleic acid sequences of theinvention is carried out by standard techniques of DNA synthesis.

In a further embodiment, the MP-protein expression vector is anexpression vector of yeast. Examples of vectors for expression in theyeast S. cerevisiae include pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88(Schultz et al. (1987) Gene 54: 113-123) and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and methods for constructingvectors which are suitable for use in other fungi such as filamentousfungi include those which are described in detail in: van den Hondel, C.A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi”, in: Applied Molecular Genetics offungi, J. F. Peberdy et al., editors, pp. 1-28, Cambridge UniversityPress: Cambridge.

As an alternative, it is possible to express the MP proteins of theinvention in insect cells using Baculovirus expression vectors.Baculovirus vectors available for the expression of proteins in culturedinsect cells (e.g. Sf9 cells) include the pAc series (Smith et al.,(1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170: 31-39).

In a further embodiment, the MP proteins of the invention may beexpressed in unicellular plant cells (such as algae) or in plant cellsof higher plants (e.g. spermatophytes such as crops). Examples ofexpression vectors of plants include those which are described in detailin Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “Newplant binary vectors with selectable markers located proximal to theleft border”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984)“Binary Agrobacterium vectors for plant transformation”, Nucl. AcidsRes. 12: 8711-8721.

In another embodiment, a nucleic acid of the invention is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). Whenused in mammalian cells, the control functions of the expression vectorare often provided by viral regulatory elements. Commonly used promotersare derived, for example, from polyoma, adenovirus2, cytomegalovirus andSimian virus 40. Other suitable expression systems for prokaryotic andeukaryotic cells can be found in Chapters 16 and 17 of Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular cloning: A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In a further embodiment, the recombinant mammalian expression vector maycause expression of the nucleic acid, preferably in a particular celltype (for example, tissue-specific regulatory elements are used forexpressing the nucleic acid). Tissue-specific regulatory elements areknown in the art. Nonlimiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43: 235-275), in particular promoters ofT-cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen andBaltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g.neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477),pancreas-specific promoters (Edlund et al., (1985) Science 230: 912-916)and mamma-specific promoters (e.g. milk serum promoter; U.S. Pat. No.4,873,316 and European Patent Application document No. 264 166).Development-regulated promoters, for example the murine hox promoters(Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546), arelikewise included.

In addition, the invention provides a recombinant expression vectorcomprising an inventive DNA molecule which has been cloned into theexpression vector in antisense direction. This means that the DNAmolecule is functionally linked to a regulatory sequence such that anRNA molecule which is antisense to MP-mRNA can be expressed (viatranscription of the DNA molecule). It is possible to select regulatorysequences which are functionally bound to a nucleic acid cloned inantisense direction and which control the continuous expression of theantisense-RNA molecule in a multiplicity of cell types; for example, itis possible to select viral promoters and/or enhancers or regulatorysequences which control the constitutive tissue-specific or celltype-specific expression of antisense RNA. The antisense expressionvector may be in the form of a recombinant plasmid, phagemid orattenuated virus, which produces antisense nucleic acids under thecontrol of a highly effective regulatory region whose activity isdetermined by the cell type into which the vector is introduced. Theregulation of gene expression by means of antisense genes is discussedin Weintraub, H. et al., Antisense-RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention relates to the host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. Naturally, these terms relate not only to a particular targetcell but also to the progeny or potential progeny of this cell. Sinceparticular modifications may appear in successive generations, due tomutation or environmental factors, this progeny is not necessarilyidentical with the parental cell but is still included in the scope ofthe term as used herein.

A host cell may be a prokaryotic or eukaryotic cell. For example, an MPprotein may be expressed in bacterial cells such as C. glutamicum,insect cells, yeast or mammalian cells (such as Chinese hamster ovary(CHO) cells or COS cells). Other suitable host cells are familiar to theskilled worker. Microorganisms which are related to Corynebacteriumglutamicum and can be used in a suitable manner as host cells for thenucleic acid and protein molecules of the invention are listed in Table3.

Conventional transformation or transfection methods can be used tointroduce vector DNA into prokaryotic or eukaryotic cells. The terms“transformation” and “transfection”, as used herein, are intended tocomprise a multiplicity of methods known in the art for introducingforeign nucleic acid (e.g. DNA) into a host cell, including calciumphosphate or calcium chloride coprecipitation, DEAE-dextran-mediatedtransfection, lipofection or electroporation. Suitable methods fortransformation or transfection of host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual. 2nd edition Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and other laboratory manuals.

In the case of stable transfection of mammalian cells, it is known that,depending on the expression vector used and transfection technique used,only a small proportion of the cells integrate the foreign DNA intotheir genome. These integrants are usually identified and selected byintroducing a gene which encodes a selectable marker (e.g. resistance toantibiotics) together with the gene of interest into the host cells.Preferred selectable markers include those which impart resistance todrugs such as G418, hygromycin and methotrexate. A nucleic acid whichencodes a selectable marker may be introduced into a host cell on thesame vector that encodes an MP protein or may be introduced on aseparate vector. Cells which have been stably transfected with theintroduced nucleic acid may be identified by drug selection (forexample, cells which have integrated the selectable marker survive,whereas the other cells die).

A homologously recombined microorganism is generated by preparing avector which contains at least one MP-gene section into which adeletion, addition or substitution has been introduced in order tomodify or functionally disrupt the MP gene. Said MP gene is preferably aCorynebacterium glutamicum MP gene, but it is also possible to use ahomolog from a related bacterium or even from a mammalian, yeast orinsect source. In a preferred embodiment, the vector is designed suchthat homologous recombination functionally disrupts the endogenous MPgene (i.e. the gene no longer encodes a functional protein; likewisereferred to as “knockout” vector). As an alternative, the vector may bedesigned such that homologous recombination mutates or otherwisemodifies the endogenous MP gene which, however, still encodes thefunctional protein (for example, the regulatory region located upstreammay be modified such that thereby the expression of the endogenous MPprotein is modified.). The modified MP-gene section in the homologousrecombination vector is flanked at its 5′ and 3′ ends by additionalnucleic acid of the MP gene, which makes possible a homologousrecombination between the exogenous MP gene carried by the vector and anendogenous MP gene in a microorganism. The length of the additionalflanking MP nucleic acid is sufficient for a successful homologousrecombination with the endogenous gene. Usually, the vector containsseveral kilobases of flanking DNA (both at the 5′ and the 3′ ends) (see,for example, Thomas, K. R. and Capecchi, M. R. (1987) Cell 51: 503 for adescription of homologous recombination vectors). The vector isintroduced into a microorganism (e.g. by electroporation) and cells inwhich the introduced MP gene has homologously recombined with theendogenous MP gene are selected using methods known in the art.

In another embodiment, it is possible to produce recombinantmicroorganisms which contain selected systems which make possible aregulated expression of the introduced gene. The insertion of an MP geneunder the control of the lac operon in a vector enables, for example,MP-gene expression only in the presence of IPTG. These regulatorysystems are known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, may be used for producing (i.e. expressing) an MPprotein. In addition, the invention provides methods for producing MPproteins by using the host cells of the invention. In one embodiment,the method comprises the cultivation of the host cell of the invention(into which a recombinant expression vector encoding an MP protein hasbeen introduced or in whose genome a gene encoding a wild-type ormodified MP protein has been introduced) in a suitable medium until theMP protein has been produced. In a further embodiment, the methodcomprises isolating the MP proteins from the medium or the host cell.

C. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors and host cells described herein may be used in one ormore of the following methods: identification of C. glutamicum andrelated organisms, mapping of genomes of organisms related to C.glutamicum, identification and localization of C. glutamicum sequencesof interest, evolutionary studies, determination of MP-protein regionsrequired for function, modulation of the activity of an MP protein;modulation of the activity of an MP pathway; and modulation-of thecellular production of a compound of interest, such as a fine chemical.The MP nucleic acid molecules of the invention have a multiplicity ofuses. First, they may be used for identifying an organism asCorynebacterium glutamicum or close relatives thereof. They may also beused for identifying C. glutamicum or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of C. glutamicum genes. Probing the extractedgenomic DNA of a culture of a uniform or mixed population ofmicroorganisms under stringent conditions with a probe which comprises aregion of a C. glutamicum gene which is unique in this organism makes itpossible to determine whether said organism is present. AlthoughCorynebacterium glutamicum itself is nonpathogenic, it is related topathogenic species such as Corynebacterium diptheriae. The detection ofsuch an organism is of substantial clinical importance.

The nucleic acid and protein molecules of the invention may serve asmarkers for specific regions of the genome. This is useful not only formapping the genome but also for functional studies of C. glutamicumproteins. The genomic region to which a particular C. glutamicumDNA-binding protein binds may be identified, for example, by cleavingthe C. glutamicum genome and incubating the fragments with theDNA-binding protein. Those fragments which bind the protein mayadditionally be probed with the nucleic acid molecules of the invention,preferably by using readily detectable labels; binding of such a nucleicacid molecule to the genomic fragment makes it possible to locate thefragment on the map of the C. glutamicum genome, and carrying out thisprocess several times using different enzymes facilitates rapiddetermination of the nucleic acid sequence to which the protein binds.Moreover, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species for thesenucleic acid molecules to serve as markers for constructing a genomicmap in related bacteria such as Brevibacterium lactofermentum.

The MP nucleic acid molecules of the invention are likewise suitable forevolutionary studies and protein structure studies. Many prokaryotic andeukaryotic cells utilize the metabolic processes in which the moleculesof the invention are involved; by comparing the sequences of the nucleicacid molecules of the invention with those sequences which encodesimilar enzymes from other organisms, it is possible to determine thedegree of evolutionary relationship of said organisms. Accordingly, sucha comparison makes it possible to determine which sequence regions areconserved and which are not, and this may be helpful in determiningthose regions of the protein, which are essential for enzyme function.This type of determination is valuable for protein engineering studiesand may give an indication as to which protein can tolerate mutagenesiswithout losing its function.

Manipulation of the MP nucleic acid molecules of the invention may causethe production of MP proteins with functional differences to wild-typeMP proteins. These proteins can be improved with respect to theirefficiency or activity, can be present in the cell in larger amountsthan normal or can be weakened with respect to their efficiency oractivity.

These changes in activity may be such that the yield, production and/orefficiency of production of one or more fine chemicals from C.glutamicum are improved. By optimizing the activity of an MP proteinwhich activates transcription or translation of a gene encoding aprotein of the biosynthesis of a fine chemical of interest or byinfluencing or deleting the activity of an MP protein which repressestranscription or translation of such a gene, it is possible to increasethe activity or activity rate of this biosynthetic pathway, owing to thepresence of increased amounts of, for example, a limiting enzyme.Correspondingly, it is possible, by modifying the activity of an MPprotein such that it constitutively inactivates posttranslationally aprotein which is involved in the degradation pathway of a fine chemicalof interest or by modifying the activity of an MP protein such that itconstitutively represses transcription or translation of such a gene, toincrease the yield and/or production rate of said fine chemical from thecell, owing to the reduced degradation of the compound.

Modulating the activity of one or more MP proteins makes it possible toindirectly stimulate the production or to improve the production rate ofone or more fine chemicals from the cell, owing to the linkage ofvarious metabolic pathways. It is possible, for example, by increasingthe yield, production and/or efficiency of production by activating theexpression of one or more enzymes in lysine biosynthesis, to increasesimultaneously the expression of other compounds such as other aminoacids which the cell usually needs in larger quantities when largerquantities of lysine are required. It is also possible to modify themetabolic regulation through in the entire cell such that the cell underthe environmental conditions of a fermentation culture (in which thesupply of nutrients and oxygen may be poor and possibly toxic wasteproducts may be present in large quantities in the environment) and mayhave improved growth or replication. Thus it is possible, for example,to improve the growth and propagation of the cells in culture, even ifthe growth conditions are suboptimal, by mutagenizing an MP proteinwhich suppresses the synthesis of molecules required for cell membraneproduction in reaction to high levels of waste products in theextracellular medium (in order to block cell growth and cell division insuboptimal growth conditions) such that said protein is no longercapable of repressing said synthesis. Such increased growth or suchincreased viability should likewise increase the yields and/orproduction rate of a fine chemical of interest from a fermentativeculture, owing to the relatively large number of cells producing thiscompound in the culture.

The abovementioned strategies for the mutagenesis of MP proteins, whichought to increase the yields of a fine chemical of interest in C.glutamicum are not intended to be limiting; variations of thesestrategies are quite obvious to the skilled worker. These strategies andthe mechanisms disclosed herein make it possible to use the nucleic acidand protein molecules of the invention in order to generate C.glutamicum or related bacterial strains expressing mutated MP nucleicacid and protein molecules so as to improve the yield, production and/orefficiency of production of a compound of interest. The compound ofinterest may be a natural C. glutamicum product which comprises the endproducts of the biosynthetic pathways and intermediates of naturallyoccuring metabolic pathways and also molecules which do not naturallyoccur in the C. glutamicum metabolism but are produced by a C.glutamicum strain of the invention.

The following examples which are not to be understood as being limitingfurther illustrate the present invention. The contents of allreferences, patent applications, patents and published patentapplications cited in this patent application are hereby incorporated byway of reference.

EXAMPLES Example 1

Preparation of Total Genomic DNA from Corynebacterium glutamicumATCC13032

A Corynebacterium glutamicum (ATCC 13032) culture was cultivated withvigorous shaking in BHI medium (Difco) at 30° C. overnight. The cellswere harvested by centrifugation, the supernatant was discarded and thecells were resuspended in 5 ml of buffer I (5% of the original culturevolume—all volumes stated have been calculated for a culture volume of100 ml). Composition of buffer I: 140.34 g/l sucrose, 2.46 g/l MgSO₄.7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 with KOH), 50ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/l MgSO₄.7 H₂O,0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/l trace elementmixture (200 mg/l FeSO₄.H₂O, 10 mg/l ZnSO₄.7 H₂O, 3 mg/l MnCl₂.4 H₂O, 30mg/l H₃BO₃, 20 mg/l CoCl₂.6 H₂O, 1 mg/l NiCl₂.6 H₂O, 3 mg/l Na₂MoO₄.2H₂O), 500 mg/l complexing agents (EDTA or citric acid), 100 ml/l vitaminmixture (0.2 ml/l biotin, 0.2 mg/l folic acid, 20 mg/l p-aminobenzoicacid, 20 mg/l riboflavin, 40 mg/l Ca pantothenate, 140 mg/l nicotinicacid, 40 mg/l pyridoxol hydrochloride, 200 mg/l myoinositol). Lysozymewas added to the suspension at a final concentration of 2.5 mg/ml. Afterincubation at 37° C. for approx. 4 h, the cell wall was degraded and theprotoplasts obtained were harvested by centrifugation. The pellet waswashed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml of TEbuffer and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5M) were added. After addition of proteinase K at a final concentrationof 200 μg/ml, the suspension was incubated at 37° C. for approx. 18 h.The DNA was purified via extraction with phenol,phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol bymeans of standard methods. The DNA was then precipitated by addition of{fraction (1/50)} volume of 3 M sodium acetate and 2 volumes of ethanol,subsequent incubation at −20° C. for 30 min and centrifugation at 12 000rpm in a high-speed centrifuge using an SS34 rotor (Sorvall) for 30 min.The DNA was dissolved in 1 ml of TE buffer containing 20 μg/ml RNase Aand dialyzed against 1000 ml of TE buffer at 4° C. for at least 3 h. Thebuffer was exchanged 3 times during this period. 0.4 ml of 2 M LiCl and0.8 ml of ethanol were added to 0.4 ml aliquots of the dialyzed DNAsolution. After incubation at −20° C. for 30 min, the DNA was collectedby centrifugation (13 000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany).The DNA pellet was dissolved in TE buffer. It was possible to use DNAprepared by this method for all purposes, including Southern blottingand constructing genomic libraries.

Example 2

Construction of Genomic Corynebacterium glutamicum (ATCC13032) Banks inEscherichia coli

Starting from DNA prepared as described in Example 1, cosmid and plasmidbanks were prepared according to known and well-established methods(see, for example, Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”. Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons).

It was possible to use any plasmid or cosmid. Particular preference wasgiven to using the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. NatlAcad. Sci. USA, 75: 3737-3741); pACYC177 (Change & Cohen (1978) J.Bacteriol. 134: 1141-1156); pBS series plasmids (pBSSK+, pBSSK− andothers; Stratagene, La Jolla, USA) or cosmids such as SuperCos1(Stratagene, La Jolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., andWaterson, R. H. (1987) Gene 53: 283-286).

Example 3

DNA Sequencing and Functional Computer Analysis

Genomic banks, as described in Example 2, were used for DNA sequencingaccording to standard methods, in particular the chain terminationmethod using ABI377 sequencers (see, for example, Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd.”, Science 269; 496-512). Sequencing primers having thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ oder5′-GTAAAACGACGGCCAGT-3′.

Example 4

In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum may be carried out bypassing a plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which cannot maintain the integrity of their geneticinformation. Common mutator strains contain mutations in the genes forthe DNA repair system (e.g., mutHLS, mutD, mutT, etc., for comparisonsee Rupp, W. D. (1996) DNA repair mechanisms in Escherichia coli andSalmonella, pp. 2277-2294, ASM: Washington). These strains are known tothe skilled worker. The use of these strains is illustrated, forexample, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.

Example 5

DNA Transfer Between Escherichia coli and Corynebacterium glutamicum

A plurality of Corynebacterium and Brevibacterium species containendogenous plasmids (such as, for example, pHM1519 or pBL1) whichreplicate autonomously (for a review see, for example, Martin, J. F. etal. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichiacoli and Corynebacterium glutamicum can be constructed readily by meansof standard vectors for E. coli (Sambrook, J. et al., (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons), to which an origin of replication for and a suitablemarker from Corynebacterium glutamicum are added. Such origins ofreplication are preferably taken from endogenous plasmids which havebeen isolated from Corynebacterium and Brevibacterium species.Particular use as transformation markers for these species are genes forkanamycin resistance (such as those derived from the Tn5 or the Tn903transposon) or for chloramphenicol (Winnacker, E. L. (1987) “From Genesto Clones—Introduction to Gene Technology”, VCH, Weinheim). There arenumerous examples in the literature for preparing a large multiplicityof shuttle vectors which are replicated in E. coli and C. glutamicum andwhich can be used for various purposes, including the overexpression ofgenes (see, for example, Yoshihama, M. et al. (1985) J. Bacteriol. 162:591-597, Martin, J. F. et al., (1987) Biotechnology, 5: 137-146 andEikmanns, B. J. et al. (1992) Gene 102: 93-98).

Standard methods make it possible to clone a gene of interest into oneof the above-described shuttle vectors and to introduce such hybridvectors into Corynebacterium glutamicum strains. C. glutamicum can betransformed via protoplast transformation (Kastsumata, R. et al., (1984)J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et al., (1989)FEMS Microbiol. Letters, 53: 399-303) and, in cases in which specificvectors are used, also via conjugation (as described, for example, inSchafer, A., et al. (1990) J. Bacteriol. 172: 1663-1666). Likewise, itis possible to transfer the shuttle vectors for C. glutamicum to E. coliby preparing plasmid DNA from C. glutamicum (by means of standardmethods known in the art) and transforming it into E. coli. Thistransformation step can be carried out using standard methods butadvantageously preferably an Mcr-deficient E. coli strain such as NM522(Gough & Murray (1983) J. Mol. Biol. 166: 1-19) is used.

Example 6

Determination of the Expression of the Mutated Protein

The observations of the activity of a mutated protein in a transformedhost cell are based on the fact that the mutated protein is expressed ina similar manner and in similar quantity to the wild-type protein. Asuitable method for determining the amount of transcription of themutated gene (an indication of the amount of mRNA available fortranslation of the gene product) is to carry out a Northern blot (see,for example, Ausubel et al., (1988) Current Protocols in MolecularBiology, Wiley: New York), with a primer which is designed such that itbinds to the gene of interest being provided with a detectable (usuallyradioactive or chemiluminescent) label such that—when the total RNA of aculture of the organism is extracted, fractionated on a gel, transferredto a stable matrix and incubated with this probe—binding and bindingquantity of the probe indicate the presence and also the amount of mRNAfor said gene. This information is an indicator of the extent to whichthe mutated gene has been transcribed. Total cell RNA can be isolatedfrom Corynebacterium glutamicum by various methods known in the art, asdescribed in Bormann, E. R. et al., (1992) Mol. Microbiol. 6: 317-326.

The presence or the relative amount of protein translated from said mRNAcan be determined by using standard techniques such as Western blot(see, for example, Ausubel et al. (1988) “Current Protocols in MolecularBiology”, Wiley, New York). In this method, total cell proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose and incubated with a probe, for example anantibody, which binds specifically to the protein of interest. Thisprobe is usually provided with a chemiluminescent or calorimetric labelwhich can be readily detected. The presence and the observed amount oflabel indicate the presence and the amount of the desired mutant proteinin the cell.

Example 7

Growth of Genetically Modified Corynebacterium glutamicum—Media andCultivation Conditions

Genetically modified corynebacteria are cultivated in synthetic ornatural growth media. A number of different growth media forcorynebacteria are known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998)Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) “TheGenus Corynebacterium”, in: The Procaryotes, Vol. II, Balows, A., etal., editors Springer-Verlag). These media are composed of one or morecarbon sources, nitrogen sources, inorganic salts, vitamins and traceelements. Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch and cellulose. Sugars may also beadded to the media via complex compounds such as molasses or otherbyproducts from sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources arealcohols and organic acids, such as methanol, ethanol, acetic acid orlactic acid. Nitrogen sources are usually organic or inorganic nitrogencompounds or materials containing these compounds. Examples of nitrogensources include ammonia gas and ammonium salts such as NH₄Cl or(NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids and complex nitrogensources such as corn steep liquor, soya meal, soya protein, yeastextracts, meat extracts and others.

Inorganic salt compounds which may be present in the media include thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid. The media usually also contain other growth factorssuch as vitamins or growth promoters, examples of which include biotin,riboflavin, thiamine, folic acid, nicotinic acid, pantothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, corn steep liquor andthe like. The exact composition of the media heavily depends on theparticular experiment and is decided upon individually for each case.Information on the optimization of media can be found in the textbook“Applied Microbiol. Physiology, A Practical Approach” (editors P. M.Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).Growth media can also be obtained from commercial suppliers, for exampleStandard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by sterile filtration. The components may be sterilizedeither together or, if required, separately. All media components may bepresent at the start of the cultivation or added continuously orbatchwise, as desired.

The cultivation conditions are defined separately for each experiment.The temperature should be between 15° C. and 45° C. and may be keptconstant or may be altered during the experiment. The pH of the mediumshould be in the range from 5 to 8.5, preferably around 7.0 and may bemaintained by adding buffers to the media. An example of a buffer forthis purpose is a potassium phosphate buffer. Synthetic buffers such asMOPS, HEPES, ACES, etc. may be used alternatively or simultaneously.Addition of NaOH or NH₄OH can also keep the pH constant duringcultivation. If complex media components such as yeast extract are used,the demand for additional buffers decreases, since many complexcompounds have a high buffer capacity. In the case of using a fermenterfor cultivating microorganisms, the pH may also be regulated usinggaseous ammonia.

The incubation period is usually in a range from several hours toseveral days. This time is selected such that the maximum amount ofproduct accumulates in the broth. The growth experiments disclosed maybe carried out in a multiplicity of containers such as microtiterplates, glass tubes, glass flasks or glass or metal fermenters ofdifferent sizes. For the screening of a large number of clones, themicroorganisms should be grown in microtiter plates, glass tubes orshaker flasks either with or without baffles. Preference is given tousing 100 ml shaker flasks which are filled with 10% (based on volume)of the required growth medium. The flasks should be shaken on an orbitalshaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Lossesdue to evaporation can be reduced by maintaining a humid atmosphere;alternatively, the losses due to evaporation should be correctedmathematically.

If genetically modified clones are investigated, an unmodified controlclone or a control clone containing the basic plasmid without insertshould also be assayed. The medium is inoculated to an OD₆₀₀ of 0.5-1.5,with cells being used which have been grown on agar plates such as CMplates (10 g/l glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5g/l yeast extract, 5 g/l meat extract, 22 g/l agar pH 6.8 with 2 M NaOH)which have been incubated at 30° C. The media are inoculated either byintroducing a saline solution of C. glutamicum cells from CM plates orby adding a liquid preculture of said bacterium.

Example 8

In Vitro Analysis of the Function of Mutated Proteins

The determination of the activities and kinetics of enzymes is wellknown in the art. Experiments for determining the activity of aparticular modified enzyme must be adapted to the specific activity ofthe wild-type enzyme, and this is within the capabilities of the skilledworker. Overviews regarding enzymes in general and also specific detailsconcerning the structure, kinetics, principles, methods, applicationsand examples of the determination of many enzyme activities can befound, for example, in the following references: Dixon, M., and Webb, E.C: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure andMechanism, Freeman, New York; Walsh (1979) Enzymatic ReactionMechanisms. Freeman, San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D:editors (1983) The Enzymes, 3rd edition, Academic Press, New York;Bisswanger, H. (1994) Enzymkinetik, 2nd edition VCH, Weinheim (ISBN3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβl, M. editors(1983-1986) Methods of Enzymatic Analysis, 3rd edition, Vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) Vol. A9, “Enzymes”, VCH, Weinheim, pp. 352-363.

The activities of proteins binding to DNA can be measured by manywell-established methods such as DNA bandshift assays (which are alsoreferred to as gel retardation assays). The action of these proteins onthe expression of other molecules can be measured using reporter geneassays (as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904and in references therein). Reporter gene assay systems are well knownand established for applications in prokaryotic and eukaryotic cells,with enzymes such as beta-galactosidase, green fluorescent protein andseveral other enzymes being used.

The activity of membrane transport proteins can be determined accordingto the techniques described in Gennis, R. B. (1989) “Pores, Channels andTransporters”, in Biomembranes, Molecular Structure and Function,Springer: Heidelberg, pp. 85-137; 199-234; and 270-322.

Example 9

Analysis of the Influence of Mutated Protein on the Production of theProduct of Interest

The effect of the genetic modification in C. glutamicum on theproduction of a compound of interest (such as an amino acid) can bedetermined by growing the modified microorganisms under suitableconditions (such as those described above) and testing the medium and/orthe cellular components for increased production of the product ofinterest (i.e. an amino acid). Such analytical techniques are well knownto the skilled worker and include spectroscopy, thin-layerchromatography, various types of coloring methods, enzymic andmicrobiological methods and analytical chromatography such as highperformance liquid chromatography (see, for example, Ullman,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III: “Product recovery and purification”, pp. 469-714, VCH:Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical Separations, in Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to measuring the end product of the fermentation, it islikewise possible to analyze other components of the metabolic pathways,which are used for producing the compound of interest, such asintermediates and byproducts, in order to determine the overallproductivity of the organism, the yield and/or the efficiency ofproduction of the compound. The analytical methods include measuring theamounts of nutrients in the medium (for example sugars, hydrocarbons,nitrogen sources, phosphate and other ions), measuring biomasscomposition and growth, analyzing the production of common metabolitesfrom biosynthetic pathways and measuring gases generated duringfermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, editors IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:0199635773) and the references therein.

Example 10

Purification of the Product of Interest from a C. glutamicum Culture

The product of interest may be obtained from C. glutamicum cells or fromthe supernatant of the above-described culture by various methods knownin the art. If the product of interest is not secreted by the cells, thecells may be harvested from the culture by slow centrifugation, and thecells may be lysed by standard techniques such as mechanial force orsonication. The cell debris is removed by centrifugation and thesupernatant fraction which contains the soluble proteins is obtained forfurther purification of the compound of interest. If the product issecreted by the C. glutamicum cells, the cells are removed from theculture by slow centrifugation and the supernatant fraction is retainedfor further purification.

The supernatant fraction from both purification methods is subjected tochromatography using a suitable resin, and either the molecule ofinterest is retained on the chromatography resin while many contaminantsin the sample are not or the contaminants remain on the resin while thesample does not. If necessary, these chromatography steps can berepeated using the same or different chromatography resins. The skilledworker is familiar with the selection of suitable chromatography resinsand the most effective application for a particular molecule to bepurified. The purified product may be concentrated by filtration orultrafiltration and stored at a temperature at which product stabilityis highest.

In the art, many purification methods are known which are not limited tothe above purification method and which are described, for example, inBailey, J. E. & Ollis, D. F. Biochemical Engineering Fundamentals,McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined bystandard techniques of the art. These techniques comprise highperformance liquid chromatography (HPLC), spectroscopic methods,coloring methods, thin-layer chromatography, NIRS, enzyme assays ormicrobiological assays. These analytical methods are compiled in: Pateket al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.(1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) BioprocessEngineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry(1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.559-566, pp. 575-581 and pp. 581-587; Michal, G. (1999) BiochemicalPathways: An Atlas of Biochemistry and Molecular Biology, John Wiley andSons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

Equivalents

The skilled worker knows, or can identify by using simply routinemethods, a large number of equivalents of the specific embodiments ofthe invention. These equivalents are intended to be included in thepatent claims below.

The information in Table 1 is to be understood as follows:

In column 1, “DNA ID”, the relevant number refers in each case to theSEQ ID NO of the enclosed sequence listing. Consequently, “5” in column“DNA ID” is a reference to SEQ ID NO:5.

In column 2, “AA ID”, the relevant number refers in each case to the SEQID NO of the enclosed sequence listing. Consequently, “6” in column “AAID” is a reference to SEQ ID NO:6.

In column 3, “Identification”, an unambiguous internal name for eachsequence is listed.

In column 4, “AA pos”, the relevant number refers in each case to theamino acid position of the polypeptide sequence “AA ID” in the same row.Consequently, “26” in column “AA pos” is amino acid position 26 of thepolypeptide sequence indicated accordingly. Position counting starts atthe N terminus with +1.

In column 5, “AA wild type”, the relevant letter refers in each case tothe amino acid, displayed in the one-letter code, at the position in thecorresponding wild-type strain, which is indicated in column 4.

In column 6, “AA mutant”, the relevant letter refers in each case to theamino acid, displayed in the one-letter code, at the position in thecorresponding mutant strain, which is indicated in column 4.

In column 7, “Function”, the physiological function of the correspondingpolypeptide sequence is listed.

One-letter code of the proteinogenic amino acids:

-   A Alanine-   C Cysteine-   D Aspartic acid-   E Glutamic acid-   F Phenylalanine-   G Glycine-   H His-   I Isoleucine-   K Lysine-   L Leucine-   M Methionine-   N Asparagine-   P Proline-   Q Glutamine-   R Arginine-   S Serine-   T Threonine-   V Valine-   W Tryptophan

Y Tyrosine TABLE 1 Genes coding for regulatory proteins DNA AA AA AA AAID: ID: Identification: pos.: wild type mutant Function: 1 2 RXA00116 26L F ASPARTATE AMINOTRANSFERASE (EC 2.6.1.1) 3 4 RXA00147 144 I LCARBAMOYL-PHOSPHATE SYNTHASE SMALL CHAIN (EC 6.3.5.5) 239 G SCARBAMOYL-PHOSPHATE SYNTHASE SMALL CHAIN (EC 6.3.5.5) 5 6 RXA00152 96 PS STOMATIN LIKE PROTEIN 237 G S STOMATIN LIKE PROTEIN 7 8 RXA00166 103 TI METHYLTRANSFERASE (EC 2.1.1.—) 9 10 RXA00278 144 G D GLUTAMINE-BINDINGPROTEIN PRECURSOR 11 12 RXA00323 41 A V GLUTAMINE SYNTHETASE (EC6.3.1.2) 80 G E GLUTAMINE SYNTHETASE (EC 6.3.1.2) 13 14 RXA00330 492 T ITHREONINE SYNTHASE (EC 4.2.99.2) 15 16 RXA00383 57 A TPROTOPORPHYRINOGEN OXIDASE (EC 1.3.3.4) 17 18 RXA00389 55 A Toxoglutarate semialdehyde dehydrogenase (EC 1.2.1.—) 481 G Eaxoglutarate semialdehyde dehydrogenase (EC 1.2.1.—) 19 20 RXA00416 223A V Hypothetical Membrane Spanning Protein 21 22 RXA00489 338 S F GMPREDUCTASE (EC 1.6.6.8) 23 24 RXA00552 295 A T THIOSULFATESULFURTRANSFERASE (EC 2.8.1.1) 25 26 RXA00579 490 P L PARA-AMINOBENZOATESYNTHASE COMPONENT I (EC 4.1.3.—) 27 28 RXA00620 141 G DPHOSPHORIBOSYLAMINOIMIDAZOLE-SUCCINOCARBOXAMIDE SYNTHASE (EC 6.3.2.6) 2930 RXA00636 146 E K Oxidoreductase FAD-binding 31 32 RXA00708 256 G S2,5-DIKETO-D-GLUCONIC ACID REDUCTASE (EC 1.1.1.—) 33 34 RXA00727 78 P SGLUTAMINE-BINDING PERIPLASMIC PROTEIN PRECURSOR 149 A VGLUTAMINE-BINDING PERIPLASMIC PROTEIN PRECURSOR 280 A VGLUTAMINE-BINDING PERIPLASMIC PROTEIN PRECURSOR 35 36 RXA00867 513 P SPOLYRIBONUCLEOTIDE NUCLEOTIDYLTRANSFERASE (EC 2.7,7.8) 37 38 RXA00955180 A V INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC4.1.1.48)/N-(5′-PHOSPHO- RIBOSYL)ANTHRANILATE ISOMERASE (EC 5.3.1.24)490 L F INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC4.1.1.48)/N-(5′-PHOSPHO- RIBOSYL)ANTHRANILATE ISOMERASE (EC 5.3.1.24) 3940 RXA00957 220 P L ANTHRANILATE SYNTHASE COMPONENT I (EC 4.1.3.27) 4142 RXA00970 133 A V HOMOSERINE KINASE (EC 2.7.1.39) 138 P S HOMOSERINEKINASE (EC 2.7.1.39) 43 44 RXA00997 117 A V 3-DEMETHYLUBIQUINONE-93-METHYLTRANSFERASE (EC 2.1.1.64) 45 46 RXA01095 262 P SINDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC 4.1.1.48) 47 48 RXA01105 270 AV HISTIDINOL-PHOSPHATE AMINOTRANSFERASE (EC 2.6.1.9) 49 50 RXA01115 72 TI 3-OXOADIPATE ENOL-LACTONE HYDROLASE (EC 3.1.1.24)/4-CARBOXY-MUCONOLACTONE DECARBOXYLASE (EC 4.1.1.44) 51 52 RXA01127 60 D N3-ISOPROPYLMALATE DEHYDROGENASE (EC 1.1.1.85) 53 54 RXA01240 23 A T GTPPYROPHOSPHOKINASE (EC 2.7.6.5) 55 56 RXA01253 112 A T COBYRIC ACIDSYNTHASE (EC 3.—.—.—) 57 58 RXA01321 156 V A FORMYLTETRAHYDROFOLATEDEFORMYLASE (EC 3.5.1.10) 59 60 RXA01381 180 G D THIF PROTEIN 61 62RXA01434 957 S F VIRULENCE FACTOR MVIN 1012 P S VIRULENCE FACTOR MVIN 6364 RXA01442 122 R H PHOSPHORIBOSYLGLYCINAMIDE FORMYLTRANSFERASE 2 (EC2.1.2.—) 65 66 RXA01483 200 R C DEOXYGUANOSINETRIPHOSPHATETRIPHOSPHOHYDROLASE (EC 3.1.5.1) 326 P S DEOXYGUANOSINETRIPHOSPHATETRIPHOSPHOHYDROLASE (EC 3.1.5.1) 67 68 RXA01515 239 A V DIHYDROPTEROATESYNTHASE (EC 2.5.1.15) 69 70 RXA01518 148 A T Hypothetical MembraneSpanning Protein 71 72 RXA01690 184 G D BRANCHED-CHAIN AMINO ACIDAMINOTRANSFERASE (EC 2.6.1.42) 73 74 RXA01746 146 G E LIPOATE-PROTEINLIGASE B (EC 6.—.—.—) 75 76 RXA01810 263 G S HEMIN-BINDING PERIPLASMICPROTEIN HMUT PRECURSOR 77 78 RXA01850 1 V M L-SERINE DEHYDRATASE (EC4.2.1.13) 45 S N L-SERINE DEHYDRATASE (EC 4.2.1.13) 79 80 RXA01940 269 AV INOSINE-URIDINE PREFERRING NUCLEOSIDE HYDROLASE (EC 3.2.2.1) 81 82RXA02021 305 S F 2,3,4,5-TETRAHYDROPYRIDINE-2-CARBOXYLATEN-SUCCINYLTRANSFERASE (HOMOLOG) 83 84 RXA02095 409 A V ABC TRANSPORTERATP-BINDING PROTEIN 914 A V ABC TRANSPORTER ATP-BINDING PROTEIN 85 86RXA02156 48 A T ACETYLGLUTAMATE KINASE (EC 2.7.2.8) 87 88 RXA02162 434 TI ARGININOSUCCINATE LYASE (EC 4.3.2.1) 89 90 RXA02176 136 P SPHOSPHOSERINE AMINOTRANSFERASE (EC 2.6.1.52) 91 92 RXA02194 129 L F ATPPHOSPHORIBOSYLTRANSFERASE (EC 2.4.2.17) 93 94 RXA02206 73 G DOXIDOREDUCTASE (EC 1.1.1.—) 114 A T OXIDOREDUCTASE (EC 1.1.1.—) 314 R COXIDOREDUOTASE (EC 1.1.1.—) 95 96 RXA02237 78 D N GUANYLATE KINASE (EC2.7.4.8) 97 98 RXA02239 168 P S PHOSPHOPANTOTHENATE--CYSTEINE LIGASE (EC6.3.2.5)/PHOSPHOPANTO- THENOYLCYSTEINE DECARBOXYLASE (EC 4.1.1.36) 99100 RXA02250 24 P S RIBX PROTEIN 101 102 RXA02295 130 G D TRANSPORTER103 104 RXA02299 36 A V ASPARTATE 1-DECARBOXYLASE (EC 4.1.1.11) 105 106RXA02382 416 E K GAMMA-GLUTAMYL PHOSPHATE REDUCTASE (GPR) (EC 1.2.1.41)107 108 RXA02390 195 G D THREONINE EFFLUX PROTEIN 109 110 RXA02499 65 GS PYRROLINE-5-CARBOXYLATE REDUCTASE (EC 1.5.1.2) 216 A TPYRROLINE-5-CARBOXYLATE REDUCTASE (EC 1.5.1.2) 111 112 RXA02516 158 P SCYSTEINE DESULFHYDRASE (EC 4.4.1.—)/SELENOCYSTEINE LYASE (EC 4.4.1.16)113 114 RXA02532 284 P S CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9) 115116 RXA02536 132 P S Carbon-nitrogen hydrolase 117 118 RXA02560 138 G EOXYGEN-INSENSITIVE NAD(P)H NITROREDUCTASE (EC 1.—.—.—)/DIHY-DROPTERIDINE REDUCTASE (EC 1.6.99.7) 192 P S OXYGEN-INSENSITIVE NAD(P)HNITROREDUCTASE (EC 1.—.—.—)/DIHY- DROPTERIDINE REDUCTASE (EC 1.6.99.7)119 120 RXA02653 448 G E pyridoxal-dependent decarboxylase 121 122RXA02754 374 P S NICOTINATE PHOSPHORIBOSYLTRANSFERASE (EC 2.4.2.11) 123124 RXA02758 110 G E PHOSPHOSERINE PHOSPHATASE (EC 3.1.3.3) 125 126RXA02790 30 L F 4-AMINO-4-DEOXYCHORISMATE LYASE (EC 4.—.—.—) 127 128RXA02912 62 G D METHYLTRANSFERASE (EC 2.1.1.—) 129 130 RXA02970 233 E KAMINOTRANSFERASE 131 132 RXA03003 209 P L ASPARTATE AMINOTRANSFERASE (EC2.6.1.1) 133 134 RXA03171 70 P S URACIL PHOSPHORIBOSYLTRANSFERASE (EC2.4.2.9) 144 L F URACIL PHOSPHORIBOSYLTRANSFERASE (EC 2.4.2.9) 199 A VURACIL PHOSPHORIBOSYLTRANSFERASE (EC 2.4.2.9) 135 136 RXA03282 312 G DCYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9) 137 138 RXA03465 224 P SMOLYBDENUM COFACTOR BIOSYNTHESIS PROTEIN A 261 P S MOLYBDENUM COFACTORBIOSYNTHESIS PROTEIN A 139 140 RXA03677 389 A T TREHALOSE/MALTOSEBINDING PROTEIN 141 142 RXA03695 70 T I METHYLTRANSFERASE (EC 2,1.1.—)143 144 RXA03754 459 G D 3-ISOPROPYLMALATE DEHYDRATASE LARGE SUBUNIT (EC4.2.1.33) 145 146 RXA03829 36 A T PROBABLE ACETOLACTATE SYNTHASE FAMILY147 148 RXA03871 33 R H MOLYBDOPTERIN BIOSYNTHESIS MOEA PROTEIN 360 T IMOLYBDOPTERIN BIOSYNTHESIS MOEA PROTEIN 149 150 RXA04120 348 G DGAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 151 152 RXA04173 41 T I GMPsynthase - Glutamine amidotransferase domain 153 154 RXA04174 856 G SACONITATE HYDRATASE (EC 4.2.1.3) 155 156 RXA04186 385 A T5-METHYLTETRAHYDROFOLATE--HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.13)492 V I 5-METHYLTETRAHYDROFOLATE--HOMOCYSTEINE METHYLTRANSFERASE (EC2.1.1.13) 990 S N 5-METHYLTETRAHYDROFOLATE--HOMOCYSTEINEMETHYLTRANSFERASE (EC 2.1.1.13) 157 158 RXA04228 279 A T GLUTAMINASE (EC3.5.1.2) 159 160 RXA04360 161 P S ANTHRANILATE PHOSPHORIBOSYLTRANSFERASE(EC 2.4.2.18) 161 162 RXA07000 398 P S GLUTAMATE SYNTHASE [NADPH] LARGECHAIN (EC 1.4.1.13) 163 164 RXA07001 110 A T MALTOSE-BINDING PROTEINPRECURSOR

1. An isolated nucleic acid molecule, coding for a polypeptide havingthe amino acid sequence in each case referred to in Table 1/column 2,wherein the nucleic acid molecule in the amino acid position indicatedin Table 1/column 4 encodes a proteinogenic amino acid different fromthe particular amino acid indicated in Table 1/column 5 in the same row.2. An isolated nucleic acid molecule as claimed in claim 1, wherein thenucleic acid molecule in the amino acid position indicated in Table1/column 4 encodes the amino acid indicated in Table 1/column 6 in thesame row.
 3. A vector, which comprises at least one nucleic acidsequence as claimed in claim
 1. 4. A host cell, which is transfectedwith at least one vector as claimed in claim
 3. 5. A host cell asclaimed in claim 4, wherein the expression of said nucleic acid moleculemodulates the production of a fine chemical from said cell.
 6. A methodfor preparing a fine chemical, which comprises culturing a cell whichhas been transfected with at least one vector as claimed in claim 3 sothat the fine chemical is produced.
 7. A method as claimed in claim 6,wherein the fine chemical is an amino acid.
 8. A method as claimed inclaim 7, wherein said amino acid is lysine.